Lyme borreliosis is now the most common arthropod-borne disease in the United States. Borrelia burgdorferi, the causative agent, has been isolated from humans, mammals, birds and arthropods and is cultivable in the laboratory. Careful structural analysis of the DNA content of several early passage isolates has revealed, in addition to a 1,000 kb linear genome, a unique mixture of terminally cross-linked linear and covalently-closed, circular DNA molecules ranging in size from 2 to 50 kilobases in length. Often DNA patterns appear to differ among isolates and to vary during laboratory passage. We have determined and compared partial 16S rRNA sequences from 23 Lyme disease spirochete isolates and aligned these with 8 sequences previously presented. The 16S rRNA signature nucleotide compositions were defined for each isolate and compared with the genomic species signature nucleotide sets previously established. Several positions were found to be variable in identity, particularly in European isolates. To identify positions truly indicative of the genomic species classification which could serve as targets for polymerase chain reaction (PCR) speciation primers, 16S rRNA based phylogenetic analyses were conducted. Based upon the identified signature nucleotides, we designed PCR primer sets which 1) amplify all spirochete species associated with Lyme disease and 2) differentiate between these species. The primer sets were tested and found to be sensitive and specific. All Lyme disease isolates tested were amplification positive. These primers should allow for the rapid speciation of isolates and for improved clinical diagnosis of Lyme disease as well. The objective of this project, therefore, is to define the genetic capacity of B. burgdorferi and other vector-borne pathogens in sufficient detail to begin the process of mapping those genes and gene products which may be important in pathogenesis.